Abstract Alzheimer's Disease (AD) is the leading cause of dementia in the United States, affecting 5.7 million Americans, yet no treatments exist. Early-onset AD is most commonly caused by familial mutations of presenilins, the catalytic subunit of the protease g-secretase. Mutated ?-secretase cleaves the amyloid precursor protein (APP) and releases toxic b-amyloid peptides associated with synapse loss. However, ?-secretase may also contribute to synaptic dysfunction in AD through mechanisms beyond APP processing. While ?-secretase contributes to AD pathology, its broader physiological roles in maintaining the proper functioning of human synapses is poorly understood. Evidence from non-neuronal cells and murine models suggest that ?-secretase may process over 90 transmembrane proteins, including synaptic signaling, scaffolding, and adhesion proteins. A knowledge gap exists on how ?-secretase maintains the proper functioning of healthy synapses in human neurons, which can further inform pathoetiologies of AD. The overall objective of this proposal is to examine in human neurons how ?-secretase regulates biochemical, morphological, and functional features of synapses, with and without chronic activity modulation. Preliminary work in human neurons validates that ?-secretase is required for b-amyloid production and cleavage of full-length APP and Neurexin (Nrxn) proteins. Early results show that ?-secretase is necessary for regulating key presynaptic and postsynaptic protein levels, as well as the number of synapses. Aim 1 will characterize the role of ?-secretase in regulating neuronal protein composition, synapse formation, and synaptic transmission to better understand its functions at human synapses. We hypothesize that ?- secretase is needed for maintaining synaptic integrity, through the processing of presynaptic and postsynaptic proteins. Aim 2 will determine how ?-secretase modulates synapses in response to chronic increases or decreases of neural activity. As other proteases have activity-dependent regulation, we hypothesize that ?- secretase regulates synaptic protein composition and synaptic transmission following chronic modulation. Understanding the role of ?-secretase at healthy synapses will provide insight into the physiological synaptic processes regulated by this protease, which will advance our understanding of the normal aging brain and AD pathology. The proposed project will establish the neuronal roles of ?-secretase in a human neuron-specific manner. Further, it will reveal the function of ?-secretase in modulating synaptic activity. Greater insight into proteolytic activity in human neurons will elucidate candidate pathways and potential therapeutic targets for AD.